Genitourinary infections affect children and adults worldwide. Gut-derived or sexually or vertically transmitted pathogens travel through the vagina to infect the female genitourinary tract. However, commensal Lactobacillus biofilms may prime genitourinary epithelial cells for anti-pathogenic responses by regulating genes which participate in natural killer T (NKT) cell activity, such as interferon gamma (IFNG) receptor 1 and the bacterial lipid antigen-presenting molecule CD1d. In turn, the formation of vaginal Lactobacillus biofilms may depend on estrogen-induced, epithelial production of glycogen, a food source for commensal-derived probiotics, "live microorganisms which when administered in adequate amounts confer a health benefit on the host". We hypothesize that estrogen stimulation of glycogen production by genitourinary epithelium promotes stability of probiotic Lactobacillus biofilms, which reciprocally modulate epithelial CD1d-mediated, IFNG-associated NKT cell responses to pathogens. This hypothesis will be tested through the following aims: Aim 1, elucidate the effects of estrogen-induced glycogen on biofilms associated with genitourinary epithelial cells;Aim 2, investigate the in vivo and in vitro mechanisms of probiotic-mediated, NKT cell interactions with genitourinary epithelium;and Aim 3, clarify the microecologic role of estrogen, genitourinary epithelial, and NKT cell interactions in shaping the genitourinary microbiome. Through these studies, the candidate seeks to become a physician investigator by complementing his background in pediatric urology and graduate work in adaptive immunology with training in biofilm microbiology and innate immunology. During the early portion of the candidate's career, the candidate will build on his foundation as a physician scientist through grantsmanship workshops;strong mentorship by experts in biofilm biology, innate immunity, and pediatric urology;scientific collaborations;practical experience;and didactic coursework that focuses on biofilms, innate immunity, and genitourinary biology. In summary, this project will lay the groundwork for a successful career as an independent investigator and will lead to the identification of novel pathways by which estrogen and probiotics protect the female genitourinary tract from infections. PUBLIC HEALTH RELEVANCE: Biofilms of commensal vaginal bacteria may have probiotic (beneficial) properties which prime the genitourinary tract for natural killer T cell responses against infections. This proposal will examine how these biofilms grow on genitourinary epithelium, and how probiotics, estrogen, genitourinary epithelium, and natural killer T cells interact to determine which bacteria grow in the female genitourinary tract.